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The Asian and Australasian Journal of Plant Science and Biotechnology ©2013 Global Science Books

Biocontrol of Cotton Damping-off Caused by Rhizoctonia solani in Salinated Soil with Rhizosphere Bacteria Dilfuza Egamberdieva* • Dilfuza Jabborova Department of Biotechnology and Microbiology, Faculty of Biology and Soil Sciences, National University of Uzbekistan, 100174 Tashkent, Uzbekistan Corresponding author: * [email protected]

ABSTRACT Pre- or post-emergence cotton seedling damping-off caused by Rhizoctonia solani is a serious problem in many cotton growing countries. Fourteen selected bacterial strains were screened for their ability to control damping-off of cotton seedlings caused by the fungus R. solani in slightly saline (EC 2.3 dS m-1) and saline (EC 7.1 dS m-1) soils. Based on the results of preliminary screening, four efficient strains, Pseudomonas alcaligenes PsA15, P. chlororaphis TSAU13, P. extremorientalis TSAU20 and Bacillus amyloliquefaciens BcA12 were selected among 14 strains. When cotton was grown in both saline soils without addition of R. solani 45% and 56% of plants were diseased in slightly saline and saline soils, respectively. In the presence of the fungal pathogen the portion of plants, which had disease symptoms, increased from 67% in slightly saline to 73% in saline soils. All bacterial strains, with the exception of B. amyloliquefaciens BcA12, showed statistically significant (P < 0.05) disease control (up to 20%) over the R. solani-infected plants grown in slightly saline soil. The higher salinity reduced the capacity of bacteria to suppress damping-off of cotton caused by R. solani. Only strain P. extremorientalis TSAU20 performed well in both saline soils. When cotton seedlings were grown in both saline field soils without addition of the fungal pathogen, all four strains showed a significant (P < 0.05) stimulatory effect on cotton dry weight (up to 57%) in comparison to the non-inoculated plants. The mechanisms, by which bacteria may use their plant-beneficial properties are also discussed. Those results showed that P. extremorientalis TSAU20 has a great biotechnological potential to stimulate plant growth and protect cotton from damping-off disease under salinated soil condition.

_____________________________________________________________________________________________________________ Keywords: biological control, cotton, cotton damping-off, Rhizoctonia solani, plant growth promotion, rhizobacteria

INTRODUCTION Salinity is a major concern for the irrigated agriculture in arid and semi-arid regions of the world (FAO 2002). Uzbekistan, located in Central Asia, is an example of a country, in which the soil salinity is a major concern. In 1990, about 48% of the total irrigated land area was suffering from the soil salinity, and by 2000, the area of salinized soil covered already 64% of the irrigated land area (Shirokova et al. 2000; Egamberdiyeva et al. 2007). Indiscriminate flood irrigation with poor drainage facilities, deep ploughing of marginal and naturally salinized soils, overexploitation of the groundwater, recycling of drainage outflows for the irrigation, and monocropping of high wateruse crops, such as cotton, are the major factors accelerating secondary soil salinization in Uzbekistan (Egamberdiyeva et al. 2007). Salt stress does not only cause a decreased metabolic activity of plant cells but it also increases susceptibility of plants towards various phytopathogens (Kurth et al. 1986; Kurth and Finkelstein 1995; Egamberdieva et al. 2010). Cotton (Gossypium hirsutum) is the largest agricultural crop in Uzbekistan. Its annual production is about 3.5-4 million tons (2007), making Uzbekistan the world’s sixth largest producer and second largest exporter of cotton (UzStat 2007). Damping-off and sore shin of cotton seedlings, killing seeds and seedlings before or after germination is a serious problem in Uzbekistan (Sheraliev et al. 2008). In Uzbekistan, estimated annual losses caused by both the soil salinity and fungus-caused root diseases are estimated to be 30%. The widespread use of fungicides has not managed to control cotton seedlings against damping-off caused by Rhizoctonia solani and other fungal pathogens (Garber et al. Received: 21 January, 2012. Accepted: 5 November, 2012.

1979; Bell 1984). According to Pimentel and Levitan (1986), a very small percentage of applied fungicides (0.1%) used for the crop protection reaches the target pathogen. Moreover, the chemicals used to protect plants against fungal pathogens are harmful to the environment. The use of, biological control agents, such as plant growthpromoting rhizobacteria (PGPR) to control plant diseases has been considered a viable alternative and environmentally friendly method (Cook and Baker 1983). PGPR are versatile micro-organisms and besides controlling plant diseases, they can increase plant growth, speed up seed germination, improve seedling emergence and protect plants from the deleterious effects of some environmental stresses, including drought and salt (Glick et al. 1998; Mayak et al. 2004; Lugtenberg and Kamilova 2004; Egamberdieva et al. 2008). Many studies have shown that microbial isolates can effectively control R. solani-induced damping off of cotton seedlings both in the laboratory and field conditions (Howell and Stipanovic 1979; Howell 1982; Hagedorn et al. 1989; Lewis and Papavizas 1991; Hagedorn et al. 1993; Zaki et al. 1998; Griffin et al. 2005; El-Sayed and Embaby 2007; Hassanin et al. 2007; Gasoni and Gurfinkel 2009). However, most studies on biological control of cotton root disease have been conducted in non-saline agricultural soils and have not addressed the problems associated with the salinity. The objective of this study was to screen and select rhizosphere bacteria, which are able both to stimulate plant growth and to control R. solani-induced damping-off of cotton seedlings growing in saline soil.

Original Research Paper

The Asian and Australasian Journal of Plant Science and Biotechnology 7 (Special Issue 2), 31-38 ©2013 Global Science Books

Table 1 Soil chemical properties of slightly saline and saline field soils used in this study. under cotton monoculture. The soil samples were taken from a depth of 0-30 cm (Egamberdieva et al. 2010) K+ Soil salinity Ca+2 Mg+2 CO32 N P CT Na+ Cl-1 -1 EC (dS m ) -------(mg kg-1)------------------------------------------------------ (g kg ) -----------------------------------------------5.92 53.4 23.7 16.1 1.06 1.30 24.8 600.2 52.0 Slightly saline (EC 2.3 dS m-1) Saline (EC 7.1 dS m-1) 6.58 67.4 24.6 17.6 0.95 1.23 24.8 813.1 94.2 Table 2 List of microorganisms used in this study. Bacterial strain Species TSAU22 Pseudomonas aureantiaca TSAU13 P. chlororaphisr TSAU6 P. extremorientalis TSAU20 P. extremorientalis TSAU1 P. putida ArG1 Arthrobacter globiformis ArS50 A. simplex ArT16 A. tumescens BcA12 Bacillus amyloliquefaciens BcP26 B. polymyxa MbP18 Mycobacterium phlei PsA15 P. alcaligenes PsD6 P. denitrificans PsM13 P. mendocina Fungi NUUF1 Rhizoktonia solani causes damping-off of cotton NUUF10 Fusarium oxysporum causes root rot of cotton TSAUF1 F. solani causes tomato foot and root rot

Origin Wheat Wheat Wheat Wheat Wheat Melon Alfalfa Alfalfa Cotton Wheat Wheat Melon Alfalfa Tomato

Reference or source Egamberdieva and Kucharova 2009 Egamberdieva and Kucharova 2009 Egamberdieva and Kucharova 2009 Egamberdieva and Kucharova 2009 Egamberdieva and Kucharova 2009 Egamberdieva and Höflich 2004 Egamberdieva and Höflich 2004 Egamberdieva and Höflich 2004 Egamberdieva and Höflich 2004 Egamberdieva and Höflich 2004 Egamberdieva and Höflich 2004 Egamberdieva and Höflich 2004 Egamberdieva and Höflich 2004 Egamberdieva and Höflich 2004

Cotton Cotton Tomato

Culture Collection of National University of Uzbekistan (CCNUU) CCNUU, Uzbekistan CCNUU, Uzbekistan

MATERIALS AND METHODS

the isolation of enhanced root tip colonizers developed by Validov et al. (2006). Briefly, wheat roots were separated from soil (10 g each) and were shaken for 1.5 h in 100 ml of phosphate buffered saline (PBS; 20 mM sodium phosphate, 150 mM NaCl, pH 7.4) and were plated on TSA/20 (1/20 of Tryptic Soya Broth with 1.5% of agar (Difco Laboratories, Detroit, MI, USA) supplemented with 1.5% NaCl. The plates were incubated at 28qC. After 48 h plates were washed with PBS. Bacterial suspensions were adjusted to an optical density of 0.1 at 620 nm (OD620 = 0.1) and were used for the inoculation of sterilized and germinated wheat seedlings. Germinated seeds were placed into the bacterial suspension with sterile forceps and shaken gently for approximately 10 min. Inoculated seeds were aseptically planted into the sand column of glass tubes of the gnotobiotic system, 5 mm below the sand surface. Six seedlings were inoculated with the bacterial suspension obtained from each sample. Subsequently, a high quality, sterilized sand (quartz sand, with particle size 0.1-0.3 mm (Wessem BV, The Netherlands) was treated with 10% Plant Nutrient Solution (PNS) (Kuiper et al. 2001). The seedlings were grown in a climatecontrolled chamber (19oV, 16/8 h day/night cycles, 70% relative humidity) for 7 days, or until the root tips penetrated the gauze. To re-isolate bacteria from the rhizosphere, the complete sand column was carefully removed from the tube. Most of the still adhering rhizosphere sand was removed from the roots and a length of 1 cm root tip was cut off with caution, in order to prevent cross-contamination from upper root parts. Root tips were shaken in 1 ml of sterile PBS and the bacterial suspension hereby obtained was diluted with PBS and plated on TSA/20 amended with 1.5% NaCl. After 48 h of incubation at 28qC, bacteria were washed from the plates with PBS and bacterial suspensions originating from the same sample were pooled together. For the inoculation of seedlings, bacterial suspensions were adjusted as it was mentioned previously. The whole cycle, starting from the inoculation of seedlings with bacterial suspension and ending to the harvest of root tips was repeated three times. After the third enrichment cycle, root colonising Pseudomonas strains were chosen for further experiments (Egamberdieva 2009; Egamberdieva and Kucharova 2009). Other strains used in this study, Arthrobacter globiformis ArG1, A. simplex ArS50; A. tumescens ArT16, P. alcaligenes PsA15, P. denitrificans PsD6, P. mendocina PsM13, Bacillus amyloliquefaciens BcA12, B. polymyxa BcP26, Mycobacterium phlei MbP18, were previously isolated from the rhizosphere of various plants grown in salinated soil using a conventional method (Egamberdieva and Hoflich 2004). For isolation of bacteria from

Study site, soil sampling and characterisation of soil Two soils, saline (EC) and slightly saline (EC) soils were sampled from an irrigated agricultural site located in Syrdarya Province (41° 00 N, 64° 00 E,) in north-eastern Uzbekistan. Both slightly saline soils and strong salinity are found at this site. According to the WRB-FAO (2006) classification, the soils of selected fields were identified as Calcisol (silt loam seirozem). The surface soil horizon was calcareous saline whereas the deeper soil horizons were only mildly alkaline (Egamberdiyeva et al. 2007). In these soils, cotton has been grown for the last 50 to 60 years under a continuous monoculture production system and under flood irrigation without proper drainage facilities but using a natural flow system. In general, high concentration of Ca2+, K+, and Na+ are associated with CO32- and Cl- ions, reflecting the dominance of carbonates and chlorides in saline soil. On average, the two kinds of soils was taken contained 42 ± 9 g of sand kg-1, 708 ± 12 g of silt kg-1, and 250 ± 13 g clay kg-1 (Egamberdieva and Kucharova 2009). Chemical properties of the two soils are shown in Table 1. The climate of the sampling site is continental, with a annual average rainfall of 200 ± 36 mm, more than 90% of the total rain falling between October and May. The average monthly minimum air temperature is 0°C in January, and the maximum one is 37°C in July. During the year, the soil temperature ranges between -2 and +35°C. The average maximum relative humidity is slightly more than 80% in January and the minimum one is less than 45% in June. Under a dry continental climate, the combination of high temperature and low rainfall during the growth season makes irrigation essential for crop production.

Plant and microorganisms used Seeds of the salt-tolerant cotton cultivar ‘Namangan’ were obtained from the Tashkent State University of Agriculture, Faculty of Plant Production. Bacterial strains used are listed in Table 2. All bacterial isolates were obtained from the Culture Collection of the National University of Uzbekistan (CCNUU). The strains Pseudomonas aureantiaca TSAU22, P. chlororaphis TSAU13, P. extremorientalis TSAU6, P. extremorientalis TSAU20, P. putida TSAU1 were previously isolated from the rhizosphere of wheat grown in salinated Uzbek soil (Egamberdieva 2009; Egamberdieva and Kucharova 2009) after using the enrichment procedure for 32

Biological control of damping off of cotton by rhizobacteria. Egamberdieva and Jabborova

the rhizosphere, 1 g of washed roots were macerated and shaken with 9 ml of sterile distilled water. The resulting suspensions were spread over the surface of a glycerol-peptone agar plate: peptone – 10 g, glycerol – 10 ml, NaCL – 5 g, KH2PO4 – 0.1 g, agar – 15 g/l sterile water. After incubation for 4 days at 28°C, the bacterial strains were isolated from the plate and identified. The fungal pathogens of cotton, R. solani, and Fusarium oxysporum and tomato root pathogen F. solani used in this study were also obtained from the CCNUU. Pseudomonas strains were cultured for 2 days for on King’s medium B (KB) (King et al. 1954) and Arthrobacter, Bacillus, and Mycobacterium strains were grown for 2 days on LC medium (containing per liter of distilled water: tryptone (Difco Laboratories) 10 g; Bacto-yeast extract (BD Biosciences) 5 g; NaCl (Sigma Chemical Co.) 10 g and agar (Difco Laboratories) 18 g) at 28°C under vigorous shaking for 2 days. The solid growth medium contained 1.8% agar (Difco Laboratories). The fungal cultures were maintained on potato dextrose agar (PDA) (Difco Laboratories) with regular sub-culturing at 1-month intervals.

were grown in pots under field conditions at 24-26°C during the day and between 12 and 14°C at night, and were watered when necessary. The number of diseased plants was determined when 40 to 60% of the fungus-infected plants but without controlling bacteria were diseased, usually 6 weeks after sowing. At harvest, plants were removed from the soil, roots were separated from shoots, were washed and examined for the symptoms of dampingoff e.g. indicated by browning and lesions of root. Roots without any disease symptoms were classified as healthy.

Plant growth promotion by bacteria The effect of the bacterial strains on the growth of cotton was measured in plastic pots containing 300 g of the weak saline and saline soil mentioned above. The inoculation treatments were setup in a randomised design with 10 replications. After six weeks of growth the root and shoot length and dry weight of the whole plants was determined.

Colonization of cotton roots by bacterial strains Preparation of fungal inoculants and infecting soils with fungi

Spontaneous and stable rifampicin (rif) (200 μg/ml) (Sigma Aldrich, St Louis, MO, USA) resistant mutants of the wild type Pseudomonas strains were used for the colonization studies. Antibiotic-resistant mutants of P. alcaligenes PsA15, P. extremorientalis TSAU20 and B. amyloliquefaciens BcA12 were selected by adding rif (200 μg/ml) at the start of exponential growth in KB agar at 28°C and by repeating the process with successive subcultures until a spontaneous mutant was selected which was resistant to rif. Prior to the root colonisation experiment, the growth rate of the mutants on KB agar (with or without rif) was compared with the growth rate of the parental strains. The cotton seeds were surface-sterilised, germinated and inoculated with rif-resistant mutant bacteria as described above. The inoculation treatments were set-up in a randomised design with 10 replications. Plants were grown in plastic pots (9 cm diameter; 12 cm deep) containing 300 g of both saline field soils under open, natural conditions in which the temperature ranged between 24 and 26°C during the day and between 10 and 12°C at night. After 6 weeks, plants were harvested and the adhering soil was removed from roots; 1 g of roots was shaken in 9 ml of sterile PBS. The resulting suspensions were evaluated for colony-forming units (cfu) according to the dilution-plate method on KB agar to which 200 μg/ml rif was added. After incubation for 2-3 days at 28°C, the bacterial colonies were enumerated and rif-resistant strains were identified for their colony characteristics (Höflich et al. 1995).

Six strains of the fungal pathogen R. solani Kuhn were used for the inoculation of soil by the method of Zheng and Sinclair (2000). Briefly, R. solani strains were cultured in potato-dextrose broth (PDB) (Difco Laboratories) in a shaker at 100 rpm. After growth for 5 days at 28°C under aeration (110 rpm), the mycelial mats were harvested by filtering culture suspensions through a single sheet of sterile filter paper (Whatman No. 1). Filter paper was dried, and the mycelial mat was weighed and homogenized in sterile distilled water. The soil was sterilized at 100°C for 24 h and mixed with the mycelial suspension using a soil mixer, to give an inoculum density of 100 mg of mycelia per kg of soil. To ensure growth of fungal strains, fungus-infected soils were kept moist for 1 week before cotton seeds were sown. To re-isolate fungal strains, a piece of root of a sick plant was removed after 5-6 weeks and plated on PDA medium in Petri dishes and incubated at 28°C under a 12-h photoperiod for 5 days. This procedure was repeated twice with each fungal strain. All six fungal strains were morphologically indistinguishable and caused similar symptoms, typical of damping off. The procedure for infecting soil with R. solani was similar to that described above, except that soils used for biological control experiment were not sterilised.

Preparation of bacterial inoculants

Antagonistic activity

Pseudomonas strains were grown overnight in KB broth King’s B medium (KB), whereas other Bacillus, Arthrobacter, and Mycobacterium strains were grown in Luria-Bertani broth (LB) (Difco Laboratories). 1.0 ml of an overnight culture was pelleted (13,000 × g) and the supernatant was discarded. Cells were washed with 1 ml of PBS and re-suspended (Leeman et al. 1995). Cell suspensions were adjusted to OD620 = 0.1 for Pseudomonas and OD620 = 0.3 for other bacterial strains, Both OD620 values corresponded to a cell density of 107 - 108 cells/ml and were used for inoculation of sterile cotton seedlings.

The antagonistic activity of bacterial strains was tested in vitro against the plant pathogenic fungi, F. oxysporum, F. solani and R. solani using a plate bioassay supplemented with 1.5% NaCl. Fungal strains were grown on PDA agar plates at 28°C for 5 days. Disks containing a fresh culture of the fungus (approx. 5 mm in diameter) were cut out of the edge of the fungal growth and placed in the centre of a 9 cm diameter Petri dish. Bacteria grown on solid LC medium, a modification of Luria broth base Miller (Difco) (containing per liter of distilled water: tryptone, 10 g; yeast extract, 5 g; NaCl, 10 g and agar-agar, 18 g) supplemented with NaCl to a final concentration of 1.5%, were streaked on the test plates perpendicular to the fungus at 2.5 cm from the disc. Plates were incubated at 28°C until the fungi had covered the control plates without bacteria (7 days). Antifungal activity was recorded as the width of the zone of growth inhibition between the fungus and the test bacterium.

Experiments for controlling damping off of cotton seedlings by bacteria in field soil Cotton seeds were surface-sterilised by immersion in 70% ethanol for 5 min and subsequently in 0.1% HgCl2 for 1 min, rinsed several times with sterile water, and allowed to germinate for 3 days at room temperature. The sterility of seeds was tested on KB agar by incubation plates for 3 days at 28°C. Seedlings were inoculated with bacteria by soaking surfacesterilised and germinated seeds in a bacterial suspension whereas uninoculated control seeds were soaked in sterile PBS buffer, both for 15 min. One seedling was planted to a plastic pot (capacity 500 ml, diameter 9 cm), containing 300 g of field soil, at a depth of approximately 1.5 cm. The treatments were arranged in a randomized complete block design with 12 replications. The plants

1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase activity 1-Amino cyclopropane-1-carboxylic acid (ACC) deaminase is an enzyme that degrades the precursor of the plant hormone ethylene, which is produced by the plant during environmental stress. Some PGPR bacteria are able to promote plant growth by lowering the endogenous ethylene synthesis in the roots through their ACC 33

The Asian and Australasian Journal of Plant Science and Biotechnology 7 (Special Issue 2), 31-38 ©2013 Global Science Books

deaminase activity (Glick et al. 1998). The method of analysing bacterial strains for their ability to use ACC as the sole nitrogen source is a trait that is a consequence of the presence of the activity of the enzyme, ACC deaminase. Synthetic Basal Medium (BM) (Lugtenberg et al. 1999) was used to check whether bacterial strains had ACC deaminase activity. BM medium was supplemented with 3.0 mM ACC (Sigma Chemical Co., St. Louis, MI, USA) (EC 4.1.99.4) and 3.0 mM (NH4)2SO4 as the sole N source (positive control) or without an added N-source (negative control).

Table 3 Controlling of damping-off of cotton by three Pseudomonas spp, and Bacillus amyloliquefaciens in slightly saline and saline soil. Slightly saline soil Saline soil Treatments Diseased plants Diseased plants (% ± SD) (% ± SD) Positive control 45 ± 3.1 56 ± 7.2 Negative control 67 ± 8.8 73 ± 10.7 P. alcaligenes PsA15 30 ± 6.0* 56 ± 5.1 P. chlororaphis TSAU13 22 ± 6.3* 46 ± 6.0 P. extremorientalis TSAU 20 20 ± 3.6* 31 ± 10.8* B. amyloliquefaciens BcA12 41 ± 9.4 55 ± 8.1

Production of indole-3-acetic acid

a

Surface-sterilizws and Pre-germinated cotton seeds were inoculated with bacteria and plants were grown under open natural conditions in pots containing slightly saline (EC value 2.3 dS m-1 ) and saline field soil (EC value 7.1 dS m-1) infected with R. solani, positive control - without R. solani, negative control – R. solani added to the soil, * Significantly different from the negative control at P < 0.05

Indole-3-acetic acid (IAA) is the most abundant endogenous auxin produced by plants, and which regulates aspects such as stem elongation and root growth. The production of IAA by bacteria is as an important factor in direct plant-growth-promoting abilities of rhizosphere bacteria (Frankenberger and Arshad 1995; Woodward and Bartel 2005). The production of IAA was determined according to the method of Bano and Musarrat (2003). Pseudomonas strains were grown in KB medium and Bacillus strain were grown in Luria–Bertani (LB) medium.medium supplemented with 1-4% NaCl with and without tryptophan (500 Pl/ml) and incubated at 28°C. After three days of cultivation, 2-ml aliquots of bacterial cultures were centrifuged at 13,000 × g for 10 min. One ml of supernatant was transferred to a fresh tube to which 100 Pl of 10 mM orthophosphoric acid and 2 ml of reagent (1 ml of 0.5 M FeCl3 in 50 ml of 35% HClO4) were added. After 25 min, the absorbance of the developed pink color was measured at 530 nm using a Perkin-Elmer Lambda 3A spectrophotometer (PerkinElmer Corp., Norwalk, CT, USA). The concentration of IAA in culture was calculated from a calibration curve of pure IAA standard.

more detailed investigations. The four strains were tested for their ability to suppress damping-off of cotton seedlings grown in slightly saline (EC 2.3 dS m-1) and saline field soils (EC 7.1 dS m-1) in order to determine how salinity affected the ability of bacterial stains to biologically control damping-off of cotton seedlings. When no R. solani was added to saline soils, the portion of diseased plants was 45% in slightly saline soil and 56% in saline soil. In the presence of the added pathogen, the portion of plants which showed disease symptoms increased to 67% in slightly saline soil and to 73% in saline soil (Table 1). In slightly saline soil, all selected bacterial isolates, with the exception of B. amyloliquefaciens BcA12, showed the capacity to control damping-off in comparison to the R. solani-infected control plants without bacteria (Table 3). In saline soil, the performance by R. solani to suppress damping-off of cotton plants was reduced. However, P. extremorientalis strain TSAU20 performed well both in slightly saline and saline soils, reducing diseased plants by as much as 20%. All four strains (P. alcaligenes PsA15 P. chlororaphis TSAU13, P. extremorientalis TSAU20, and B. amyloliquefaciens BcA12) significantly (P < 0.05) increased the length and dry weight of cotton roots and shoots in both saline soils relative to uninoculated control plants (Table 4). Nevertheless, the stimulation of growth of cotton plants by bacterial strains was higher in saline soil than in slightly saline soil. Again, the best performer was strain P. extremorientalis TSAU20, which increased the root and shoot length by 67% and their dry weight by 47% (Table 4). P. alcaligenes PsA15, P. chlororaphis TSAU13, P. extremorientalis TSAU20 and B. amyloliquefaciens BcA12 were tested for their ability to colonize cotton roots when grown in slightly saline and in saline soil. This experiment was performed by using rif-resistant mutants of the four parental strains. All four bacterial strains were able to survive in the rhizosphere of two 2-month-old cotton plants (Table 5). The population of the rif-resistant mutants was log 4.01 CFU/g cotton roots in slightly saline soil and log 3.42 CFU/g cotton roots of cotton grown in saline soil. Among the strains tested, P. extremorientalis TSAU20 showed the highest ability to colonise the rhizosphere of cotton grown in slightly saline and saline soil. The production of auxin (IAA) of the strains P. alkaligenes PsA15, P. chlororaphis TSAU13, P. extremorientalis TSAU20 and B.amyloliquefaciens BcA12 was tested by growing in KB medium under saline condition (1-4% NaCl). All strains tested appeared to produce IAA in media containing up to 3% NaCl (Table 5). The presence of tryptophan stimulated auxin production in P. alcaligenes PsA15 (up to 71%), P. chlororaphis TSAU13 (80%), P. extremorientalis TSAU20 (97%) and B.amyloliquefaciens BcA12 (56%). When four strains were tested for their antagonistic activity towards F. oxysporum, F. solani and R. solani, only P. chlororaphis TSAU13 was antagonist against all tested pathogenic fungi (Table 5). This strain was also able to

Production of cell wall degrading enzymes The production of lytic enzymes such as lipase, cellulase, protease and glucanase by rhizosphere microorganisms can result in the direct suppression of plant pathogenic fungi (Lugtenberg et al. 2001). Lipase (EC 3.1.1.3) activity of bacterial strains was determined by the Tween lipase indicator assay according to Howe and Ward (1976). Pseudomonas strains were grown on KB agar and the Bacillus strain was grown on LB agar supplemented with 1.5% NaCl and containing 2% Tween-80 at 28°C After 5 days, the degradation of Tween by lipase was detected as a clear halo around the bacterial inoculum. Protease secretion was revealed by growing strains on agar plates described above but amended with skimmed milk to a final concentration of 5%. After 1-2 days, the halo appearing around bacterial colonies indicated the presence of extracellular protease (Brown and Foster 1970). Cellulase (EC 3.2.1.4) activity was detected using the substrate carboxymethylcellulose (Sigma Aldrich) in top-agar plates (Hankin and Anagnostakis 1977).

Statistical procedures Data were tested for statistical significance using the analysis of variance package included in Microsoft Excel 98. Comparisons were done using Student’s t-test. Mean comparisons were conducted using a least significant difference (LSD) test (P = 0.05).

RESULTS The fourteen bacterial strains were evaluated for their ability to control damping-off of cotton seedlings caused by the fungus R. solani and to promote plant growth in slightly saline (EC 2.3 dS m-1) and saline (EC 7.1 dS m-1) soils (Table 1). The bacterial candidates were previously isolated from the rhizosphere of various plants. Based on the results of a preliminary screening experiment, four efficient PGPR strains namely, P. alcaligenes PsA15 P. chlororaphis TSAU13, P. extremorientalis TSAU20, and B. amyloliquefaciens BcA12 showed statistically significant (P < 0.05) capacity to control dampingoff in comparison to the R. solani-infected control plants without added bacteria. These four strains were selected for 34

Biological control of damping off of cotton by rhizobacteria. Egamberdieva and Jabborova

Table 4 Promotion of plant growth of cotton by three Pseudomonas spp. and Bacillus amyloliquefaciens in slightly saline and saline field soil. Slightly saline soil Saline soil Treatmentsa Root length, cm Shoot length, cm Dry matter Root length, cm Shoot length, cm Dry matter g/plant g/plant Control 100 (7.4) b 100 (7.3) b 100 (0.760) c 100 (6.4) b 100 (5.9) b 100 (0.57) c P. alcaligenes PsA15 136* 125* 119 155* 159* 157* P. chlororaphis TSAU13 136* 135* 144* 124 149* 146* P. extremorientalis TSAU20 153* 143* 144* 142* 167* 147* B.amyloliquefaciens BcA12 146* 145* 128* 145* 161* 136* a Pre-germinated cotton seeds were coated with bacteria, and plants were grown under open natural conditions in pots containing slightly saline (EC value 2.3 dS m-1 ) and saline field soil (EC value 7.1 dS m-1), * Significantly different from the control at P < 0.05

+ -

+ -

+ -

+ -

Cellulase

Protease

Lipase

F. oxysporum

R. solani + -

+ + -

3% NaCl

-

2 % NaCl

+ -

1 % NaCl

P. alcaligenes PsA15 P. chlororaphis TSAU13 P. extremorientalis TSAU20 B. amyloliquefaciens BcA12

R. solani

HCN

ACC deaminase

Table 5 Traits possibly involved in biocontrol and/or plant growth-promoting activity of bacterial strains Antagonistic activity Production of Production of IAAb Strains a a against exoenzymes

2.4 8.5 7.4 6.7

10.0 7.8 6.0 6.3

8.5 7.1 4.7 6.0

Bacterial colonization (Log (CFU)/g root-1)c Slightly Saline saline

3.82 ± 0.12 3.80 ± 0.13 4.01 ± 0.18 3.68 ± 0.10

3.71 ± 0.22 3.61 ± 0.18 3.76 ± 0.10 3.42 ± 0.14

a) All tests conducted with addition of 1.5%NaCl b) Auxin (IAA) level in μg/ml of after 5 days of incubation at 28°C. Pseudomonas strains were grown in KB medium and Bacillus strain were grown in Luria–Bertani (LB) medium supplemented with 1-4% NaCl. c) The number of bacteria in the rhizosphere of cotton grown in slightly and saline field soil for 2 months

ping-off cucumber (Cucumis sativus) seedlings (Jung et al. 2003), potato (Solanum tuberosum) (Brewer and Larkin 2005) and tomato (Lycopersicon esculentum) (Yangui et al. 2008) caused by R. solani. Patil et al. (2011) found that antagonistic actinomycetes could biologically control R. solani in tomato under greenhouse conditions. However, these strains were used as biological control agents under favourable soil conditions. The negative effect of abiotic factors on the biological control ability of bacterial strains was reviewed by Burpee (1990) and Kalifa et al. (2012). These factors include temperature, pH, moisture, soil type, inorganic and organic constitutes, but the problem associated with salinity was not addressed. Rangarajan et al. (2003) studied antagonistic Pseudomonas strains that were screened for in vitro antibiosis against Xanthomonas oryzae pv. oryzae and R. solani – the bacterial leaf blight (BB) and sheath blight (ShB) pathogens of rice (Oryza sativa), respectively. They suppressed disease by 46 to 82% under saline soil condition. In our study we observed that higher saline soil may inhibit the ability of bacterial strains to control damping-off of cotton. Salt stress does not only cause a decline in the metabolic activity of plant cells but it also results in an increased susceptibility of plants towards phytopathogens (Kurth et al. 1986; Werner and Finkelstein 1995). This result is consistent with our finding that a higher percentage of diseased plants (56%) were observed in saline soil compared to plants grown in slightly saline soil (45%) and that were not infested with R. solani. All four selected strains, with the exception of B. amyloliquefaciens BcA12, showed significant (P < 0.05) repression of damping-off of cotton caused by R. solani in slightly saline soil relative to the Fusarium-infected control plants. In saline soil they were not effective, whereas only one strain, P. extremorientalis TSAU20, was able to control dampingoff of cotton in saline soil.

produce fungal cell wall-degrading enzymes, namely lipase, protease, cellulase and HCN. The remaining strains, i.e., P. alcaligenes PsA15, P. extremorientalis TSAU20 and B. amyloliquefaciens BcA12, did not show antagonistic activity. They were also negative for HCN production and lacked fungal cell wall-degrading enzyme activities except for P. extremorientalis strain TSAU20, which was able to produce cellulase (Table 5). Only strain P. alcaligenes PsA15 could utilize ACC as the sole N source, indicating the presence of ACC deaminase, which plays a role in reducing the levels of the stress compound ethylene in plants. Other strains were negative the ACC deaminase activity (Table 5). DISCUSSION Biological control of damping-off of cotton by rhizosphere bacteria This is the first report of the use of bacterial strains to control damping-off of cotton caused by R. solani in salinated soil. Four of the selected bacterial strains tested in this study significantly (P < 0.05) reduced the damping-off of cotton caused by R. solani in saline soil. The bacterial strains P. alcaligenes PsA15 and B. amyloliquefaciens BcA12, known to reduce Vertciullium wilt of cotton (Egamberdiyeva 2003), and two other strains, P. chlororaphis TSAU13 and P. extremorientalis TSAU20, were isolated from the rhizosphere of wheat grown in salinated Uzbek soil after using an enrichment procedure for the isolation of enhanced root tip colonizers. Biological control of damping-off of cotton caused by R. solani using PGPR was observed by other authors (Wather and Gindrat 1988; Alagesaboopathi 1994; Hassanin et al. 2007). The beneficial effects on cotton stand were also obtained with seed treatments of Trichoderma viride, T. harzianum (Sivan and Chet 1986; De Vay et al. 1987), Gliocladium roseus (Howell 1982), Coniothyrium minitans (ElSayed and Embaby 2007) and Burkholderia cepacia (Heydari and Misagli 1998). It has been also reported that a number of microbial isolates have proven to be an effective biocontrol agent against damping-off of various crops. Mavrodi et al. (2012) studied the suppression of Rhizoctonia disease in wheat by Pseudomonas strains. Other authors observed that PGPR suppress symptoms of dam-

Plant growth promotion by rhizosphere bacteria In the absence of a pathogen, all four strains significantly (P < 0.05) increased root and shoot growth and dry weight of cotton in both slightly saline and saline soil. Plant growth was higher in saline soil than in uninoculated control plants. In previous studies, Rashid et al. (2000), Hafeez et al. (2002) and Anjum et al. (2007) observed that bacterial inoculum significantly increased seed cotton yield, and 35

The Asian and Australasian Journal of Plant Science and Biotechnology 7 (Special Issue 2), 31-38 ©2013 Global Science Books

plant height over their respective controls under normal soil conditions. Similar results were reported by Yue et al. (2007) for Klebsiella oxytoca, which was able to relieve salt stress and promote the growth of cotton seedlings in salinated soil. After treatment with bacterial strains, plant height and dry weight of cotton increased by 14.9 and 26.9%, respectively, compared to the control. In K-deficient soil, Bacillus edaphicus also stimulated growth of cotton and the N and P content of above-ground plant components (Sheng 2005). According to Paula et al. (1992), the magnitude of a plant’s response to any microbial inoculation can be greatly affected by the soil condition. The greatest benefits occurred when crops encountered stressful conditions (Lazarovits and Nowak 1997); for example, high pH makes nutrients less available to plants. Bacterial inoculation stimulated taproot growth and increased the number of lateral roots which may result in better absorption of water and nutrients from the soil (Hoflich and Kuhn 1996). Similar results we observed in our previous work (Egamberdiyeva 2007) in which in nutrient-poor arid soil, bacterial strains significantly (P < 0.05) enhanced early plant growth of maize, and the inoculation could compensate for nutrient deficiency and improve plants’ development.

nisms the observed biological control of damping-off of cotton and plant growth stimulation in saline soil can be based. Some mechanisms such as antagonism (Kuiper et al. 2001; Bloemberg and Lugtenberg 2004; Lugtenberg and Kamilova 2009), competition for nutrients and niches (Kamilova et al. 2005) and production of phytohormones (Frankenberger and Arshad 1995; Spaepen et al. 2009) were demonstrated. Only strain P. chlororaphis TSAU13 was antagonistic towards pathogenic fungi F. oxysporum, F. solani and R. solani under laboratory conditions. Safiyazov et al. (1995) reported antagonistic activity of bacterial strains suppressing the development of cotton seedling disease. The potential root colonising bacteria P. extremorientalis strain TSAU20 did not show antagonistic activity against F. oxysporum, F. solani and R. solani even though this strain was able to control damping-off of cotton in both saline soils. In our previous work, this strain was also able to control cucumber foot and root rot through competition for nutrients and niches (Egamberdieva et al. 2010). Thus, our result suggests that competition for nutrients and niches might be an important factor in controlling plant diseases in saline soils. Wang et al. (2004) also observed that P. fluorescens CS85, which did not inhibit the growth of several fungal pathogens in the laboratory, was an effective biocontrol agent against cotton seedling diseases through competition for nutrient and niches. All four bacterial strains tested were able to produce IAA under saline condition (Egamberdieva 2009). It is known that unfavourable environmental factors such as salinity and drought cause sharp changes in the balance of phytohormones associated with a decline in the level of growth-activating hormones such as IAA (Zholkevich and Pustovoytova 1993; Jackson 1997; Sakhabutdinova et al. 2003). In such a condition, the IAA-producing bacteria may supply additional phytohormones to the plant, thus may help stimulate root growth and reverse the growth-inhibiting effect of salt stress to a certain extent in both shoot and root growth (Kabar 1987; Afzal et al. 2005). Strain P. alcaligenes PsA15 was able to utilize ACC as the sole nitrogen source. This suggests that strain synthesize ACC deaminase, which can cleave the plant ethylene precursor ACC, and thereby lower the level of ethylene in a developing or stressed plant (Glick et al. 1998; Penrose et al. 2001; Glick 2005). Yue et al. (2007) demonstrated that Klebsiella oxytoca strain Rs-5, which had ACC deaminase activity, could relieve salt stress and promote cotton seedling growth more in saline than in non-saline soil. The differential effects of salinity to bacterial control of cotton disease, as shown in this study, may explain importance of the selection of isolates whose biocontrol activity is not adversely affected by an increase in salinity. It is possible to recommend selected enhanced root-colonizing P. extremorientalis strain TSAU20 bacteria to control damping-off of cotton caused by R. solani and to stimulate plant growth under conditions of soil salinity.

Root colonisation and survival of bacterial strains Inoculation of plants with PGPR will not result in significant effects unless the environment supports growth and survival of the introduced microorganisms in a highly competitive environment (Wessendorf and Lingens 1989; Van Elsas and Heijnen 1990; Devliegher et al. 1995). Lugtenberg et al. (1999) and Thomashow and Weller (1995) reported that competitive root colonisation by rhizosphere bacteria is considered to be one of the mechanisms of biological control of root disease by PGPR. The successful colonization of the rhizosphere by introduced beneficial bacteria usually requires that the bacteria not only be welladapted to the rhizosphere, but that it also have some selective advantage over numerous indigenous bacteria with the potential to colonize that rhizosphere (Kawaguchi et al. 2003). The survival and growth of the inoculated bacteria in soil largely depends on the availability of empty niches, and the capacity of competing with the better adapted native microflora (Rekha et al. 2007). In our study the rif-resistant mutants of four effective strains were able to colonize the rhizosphere of cotton due to their persistence in saline soil. The strain P. extremorientalis TSAU20, which was isolated as an enhanced wheat root colonizer (Egamberdieva and Kucharova 2009) showed higher colonisation rate in the rhizosphere of cotton, whereas B. amyloliquefaciens BcA12 was demonstrated to have lower colonization ability. This is consistent with data on root colonization by PGPR in the root systems of corn, tomato, broad bean, barley, and canola (Shaw et al. 1992; Ramos et al. 2000). Pseudomonas fluorescens CS85, which was previously isolated from the rhizosphere of cotton seedlings, acts as both a PGPR and as a biocontrol agent against cotton pathogens, including R. solani, Fusarium oxysporum f sp. vasinfectum, and Verticillium dahlia which colonize all surfaces of the young plant root zones, such as roots hairs and lateral roots during the period of plant growth (Wang et al. 2004). Factors that can influence the survival of microorganisms in soil include soil-type, condition, pH, temperature, water potential as well as the presence of other soil organisms (Benizri et al. 2001). According to Diby et al. (2005), root colonisation potential of a strain was not hampered by higher salinity of soil. The strains used in this study were able to colonize the rhizosphere of cotton due to their competitiveness and persistence in saline soil.

ACKNOWLEDGEMENTS This study was supported by the Academy of Sciences for the Developing World (TWAS 07-271RG/BIO/AS). The authors thank Leena Rasanen and Dr. Jaime A. Teixeira da Silva for significant improvements to grammar.

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